PNA probes, probe sets, methods and kits pertaining to the detection of Candida

ABSTRACT

This invention is related to novel PNA probes, probe sets, methods and kits pertaining to the detection of one or more species of  Candida  yeast. Non-limiting examples of probing nucleobase sequences that can be used for the probes of this invention can be selected from the group consisting of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7), CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8), GAC-GCC-AAA-GAC-GCC (Seq. Id. No. 9), ATC-GTC-AGA-GGC-TAT-AA (Seq. Id. No. 10), TAG-CCA-GAA-GAA-AGG (Seq. Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id. No. 12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13), TCC-CAG-ACT-GCT-CGG (Seq. Id. No. 14), TCC-AAG-AGG-TCG-AGA (Seq. Id. No. 15), GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16), GCC-GCC-AAG-CCA-CA (Seq. Id. No. 17), GGA-CTT-GGG-GTT-AG (Seq. Id. No. 18), CCG-GGT-GCA-TTC-CA (Seq. Id. No. 19), ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20), GAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21), CTG-GTT-CGC-CAA-AAA-G (Seq. Id. No. 22) and AGT-ACG-CAT-CAG-AAA (Seq. Id. No. 23).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/207,283, filed Aug. 20, 2011, now U.S. Pat. No. 8,912,312, which is acontinuation of U.S. application Ser. No. 10/150,045, filed May 17,2002, now U.S. Pat. No. 8,026,051, which claims the benefit of U.S.Provisional Application No. 60/292,147 filed on May 18, 2001; each ofwhich disclosures are herein incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The US Government has certain rights in this invention as provided forby the terms of the Cooperative Research and Development Agreement(CRADA) No. 58-3K95-8-631 by and between Boston Probes, Inc. and theUnited States Department of Agriculture.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is related to the field of probe-based detection,analysis and/or quantitation of microorganisms. More specifically, thisinvention relates to novel PNA probes, probe sets, methods and kitspertaining for the detection, identification and/or enumeration oforganisms of the various species of the Candida genus.

2. Description of the Related Art

Nucleic acid hybridization is a fundamental process in molecularbiology. Probe-based assays are useful in the detection, quantitationand/or analysis of nucleic acids. Nucleic acid probes have long beenused to analyze samples for the presence of nucleic acid from bacteria,fungi, virus or other organisms and are also useful in examininggenetically-based disease states or clinical conditions of interest.Nonetheless, probe-based assays have been slow to achieve commercialsuccess. This lack of commercial success is, at least partially, theresult of difficulties associated with specificity, sensitivity andreliability.

Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, anucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is anon-naturally occurring polyimide that can hybridize to nucleic acid(DNA and RNA) with sequence specificity (See: U.S. Pat. No. 5,539,082and Egholm et al., Nature 365: 566-568 (1993)). Being a non-naturallyoccurring molecule, unmodified PNA is not known to be a substrate forthe enzymes that are known to degrade peptides or nucleic acids.Therefore, PNA should be stable in biological samples, as well as have along shelf-life. Unlike nucleic acid hybridization, which is verydependent on ionic strength, the hybridization of a PNA with a nucleicacid is fairly independent of ionic strength and is favored at low ionicstrength, conditions that strongly disfavor the hybridization of nucleicacid to nucleic acid (Egholm et al., Nature, at p. 567). The effect ofionic strength on the stability and conformation of PNA complexes hasbeen extensively investigated (Tomac et al., J. Am. Chem. Soc. 118:5544-5552 (1996)). Sequence discrimination is more efficient for PNArecognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, atp. 566). However, the advantages in point mutation discrimination withPNA probes, as compared with DNA probes, in a hybridization assay,appears to be somewhat sequence dependent (Nielsen et al., Anti-CancerDrug Design 8:53-65, (1993) and Weiler et al., Nucl. Acids Res. 25:2792-2799 (1997)).

Though they hybridize to nucleic acid with sequence specificity (See:Egholm et al., Nature, at p. 567), PNAs have been slow to achievecommercial success at least partially due to cost, sequence specificproperties/problems associated with solubility and self-aggregation(See: Bergman, F., Bannwarth, W. and Tam, S., Tett. Lett. 36:6823-6826(1995), Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P. E., Angew.Chem. Int. Ed. Engl. 35:1939-1942 (1996) and Lesnik, E., Hassman, F.,Barbeau, J., Teng, K. and Weiler, K., Nucleosides & Nucleotides16:1775-1779 (1997) at p 433, col. 1, ln. 28 through col. 2, ln. 3) aswell as the uncertainty pertaining to non-specific interactions thatmight occur in complex systems such as a cell (See: Good, L. et al.,Antisense & Nucleic Acid Drug Development 7:431-437 (1997)). However,problems associated with solubility and self-aggregation have beenreduced or eliminated (See: Gildea et al., Tett. Lett, 39: 7255-7258(1998)). Nevertheless, their unique properties clearly demonstrate thatPNA is not the equivalent of a nucleic acid in either structure orfunction. Consequently, PNA probes should be evaluated for performanceand optimization to thereby confirm whether they can be used tospecifically and reliably detect a particular nucleic acid targetsequence, particularly when the target sequence exists in a complexsample such as a cell, tissue or organism.

SUMMARY OF THE INVENTION

This invention is directed to PNA probes, probe sets, methods and kitsuseful for detecting, identifying and/or quantitating Candida yeast in asample. The PNA probes, probe sets, methods and kits of this inventionare suitable for the analysis of nucleic acid, whether or not it ispresent within an organism of interest. Accordingly, this invention canbe used for both the analysis of organisms or for the analysis ofnucleic acid extracted from or derived from an organism of interest.

Generally, this invention can be particularly useful for thedetermination of particular species of the Candida genus. The PNA probesand probe sets of this invention comprise probing nucleobase sequencesthat are particularly useful for the specific detection of certainspecies of Candida, including C. albicans (also comprising C.stellatoidea, a biovar of C. albicans), C. dubliniensis, C. krusei, C.glabrata, C. parapsilosis and C. tropicalis. A particularly usefulprobing nucleobase sequence is Seq. Id. No. 1 (See: Table 1) because itcan be highly specific for C. albicans, a pathogen that is particularlyimportant to determine as early as possible in the area of blood cultureanalysis. Exemplary probing nucleobase sequences for the probes of thisinvention are listed in Table 1, below. The species of Candida for whicheach probe is intended to determine is also listed in the Table.

This invention is further directed to a method suitable for detecting,identifying and/or quantitating a species of Candida in a sample. Forexample, the method can be directed to the detection of a particularspecies of Candida wherein the species is selected from the groupconsisting of: C. albicans, C. dubliniensis, C. krusei, C. glabrata, C.parapsilosis and C. tropicalis.

The method can comprise contacting the sample with one or more PNAprobes, wherein suitable probes are described herein. According to themethod, the presence, absence and/or number of the one or more speciesof Candida in the sample is then detected, identified and/orquantitated. Detection, identification and/or quantitation is madepossible by correlating the hybridization, under suitable hybridizationconditions or suitable in-situ hybridization conditions, of the probingnucleobase sequence of a PNA probe to the target sequence with thepresence, absence and/or quantity of target organism in the sample. Thiscorrelation is made possible by direct or indirect determination of theprobe/target sequence hybrid.

In yet another embodiment, this invention is directed to kits suitablefor performing an assay that determines the presence, absence and/orquantity of a species of Candida in a sample. The kits of this inventioncomprise one or more PNA probes and other reagents or compositions thatare selected to perform an assay or otherwise simplify the performanceof an assay.

The PNA probes, probe sets, methods and kits of this invention have beendemonstrated to be relatively specific for the species of Candida forwhich they are intended to determine. Moreover, the assays describedherein are rapid (2-3 hours or less), sensitive, reliable and capable,in a single assay, of identification as well as detection and/orenumeration of the organisms listed in Table 1.

The PNA probes, probe sets, methods and kits of this invention can beparticularly useful for the determination of Candida in food, beverages,water, pharmaceutical products, personal care products, dairy productsand/or environmental samples. The analysis of beverages includes soda,bottled water, fruit juice, beer, wine or liquor products. Suitable PNAprobes, probe sets, methods and kits can be particularly useful for theanalysis of raw materials, equipment, products or processes used tomanufacture or store food, beverages, water, pharmaceutical products,personal care products dairy products or for the analysis ofenvironmental samples.

Additionally, the PNA probes, probe sets, methods and kits of thisinvention can be particularly useful for the detection of Candidaspecies in clinical samples and clinical environments. By way of anon-limiting example, the PNA probes, probe sets, methods and kits ofthis invention can be particularly useful in the analysis of bloodcultures or in samples derived therefrom (e.g. subcultures). Othernon-limiting examples of clinical samples include: sputum, laryngealswabs, gastric lavage, bronchial washings, biopsies, aspirates,expectorates, body fluids (e.g. spinal, pleural, pericardial, synovial,blood, pus, amniotic, and urine), bone marrow and tissue sections(including cultures and subcultures derived therefrom). Suitable PNAprobes, probe sets, methods and kits will also be particularly usefulfor the analysis of clinical specimens, equipment, fixtures or productsused to treat humans or animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises 5 images that were obtained using a fluorescentmicroscope using a FITC/Texas red filter for five culture samples, eachculture sample representing a different species of Candida, wherein allsamples have been treated with the PNA probe Can26S03 having sequence IdNo. 1 as the probing nucleobase sequence. Only the sample containing C.albicans is positive thereby demonstrating that the probe is highlyspecific for C. albicans and can be used to distinguish numerous otherCandida species, including the difficult to distinguish, C.dubliniensis.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions:

-   a. As used herein, “nucleobase” means those naturally occurring and    those non-naturally occurring heterocyclic moieties commonly known    to those who utilize nucleic acid technology or utilize peptide    nucleic acid technology to thereby generate polymers that can    sequence specifically bind to nucleic acids. Non-limiting examples    of suitable nucleobases include: adenine, cytosine, guanine,    thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,    5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine,    2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),    hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and    N8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitable    nucleobase include those nucleobases illustrated in FIGS. 2(A) and    2(B) of Buchardt et al. of U.S. Pat. No. 6,357,163 (incorporated    herein by reference).-   b. As used herein, “nucleobase sequence” means any segment, or    aggregate of two or more segments of a polymer that comprises    nucleobase-containing subunits. Non-limiting examples of suitable    polymers include oligodeoxynucleotides (e.g. DNA),    oligoribonucleotides (e.g. RNA), peptide nucleic acids (PNA), PNA    chimeras, PNA oligomers, nucleic acid analogs and/or nucleic acid    mimics.-   c. As used herein, “target sequence” is a nucleobase sequence of a    polynucleobase strand sought to be determined. The target sequence    can be a subsequence of the rRNA of Candida yeast.-   d. As used herein, “polynucleobase strand” means a complete single    polymer strand comprising nucleobase subunits.-   e. As used herein, “nucleic acid” is a nucleobase    sequence-containing polymer, or polynucleobase strand, having a    backbone formed from nucleotides, or analogs thereof. Preferred    nucleic acids are DNA and RNA. For the avoidance of any doubt, PNA    is a nucleic acid mimic and not a nucleic acid analog.-   f. As used herein, “peptide nucleic acid” or “PNA” means any    oligomer or polymer segment comprising two or more PNA subunits    (residues), including, but not limited to, any of the oligomer or    polymer segments referred to or claimed as peptide nucleic acids in    U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,    5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459,    5,891,625, 5,972,610, 5,986,053, 6,107,470 and 6,357,163; all of    which are herein incorporated by reference. The term “peptide    nucleic acid” or “PNA” shall also apply to any oligomer or polymer    segment comprising two or more subunits of those nucleic acid mimics    described in the following publications: Lagriffoul et at,    Bioorganic & Medicinal Chemistry Letters, 4: 1081-1082 (1994);    Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6:    793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478 (1996);    Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et.    al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett.    Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem.    Lett. 4: 1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal    Chemistry Letters, 7:1743-1746 (1997); Lowe et al., J. Chem. Soc.    Perkin Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc.    Perkin Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin.    Trans. 1 1:5 55-560 (1997); Howarth et al., J. Org. Chem. 62:    5441-5450 (1997); Altmann, K-H et al., Bioorganic & Medicinal    Chemistry Letters, 7: 1119-1122 (1997); Diederichsen, U., Bioorganic    & Med. Chem. Lett., 8: 165-168 (1998); Diederichsen et al., Angew.    Chem. Int. Ed., 37: 302-305 (1998); Cantin et al., Tett. Lett., 38:    4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176    (1997); Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997); Kumar    et at, Organic Letters 3(9): 1269-1272 (2001); and the Peptide-Based    Nucleic Acid Mimics (PENAMs) of Shah et al. as disclosed in    WO96/04000.

In certain embodiments, a “peptide nucleic acid” or “PNA” is an oligomeror polymer segment comprising two or more covalently linked subunits ofthe formula:

wherein, each J is the same or different and is selected from the groupconsisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. Each K isthe same or different and is selected from the group consisting of O, S,NH and NR¹. Each R′ is the same or different and is an alkyl grouphaving one to five carbon atoms that may optionally contain a heteroatomor a substituted or unsubstituted aryl group. Each. A is selected fromthe group consisting of a single bond, a group of the formula;—(CJ₂)_(s)- and a group of the formula; —(CJ₂)_(s)C(O)—, wherein, J isdefined above and each s is a whole number from one to five. Each t is 1or 2 and each u is 1 or 2. Each L is the same or different and isindependently selected from: adenine, cytosine, guanine, thymine,uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine), other naturally occurring nucleobase analogsor other non-naturally occurring nucleobases.

In certain other embodiments, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to theN-α-glycine nitrogen of the N-[2-(aminoethyl)]glycine backbone through amethylene carbonyl linkage; this currently being the most commonly usedform of a peptide nucleic acid subunit.

-   g. As used herein, the terms “label”, “reporter moiety” or    “detectable moiety” are interchangeable and refer to moieties that    can be attached to PNA oligomer or antibody, or otherwise be used in    a reporter system, to thereby render the oligomer or antibody    detectable by an instrument or method. For example, a label can be    any moiety that: (i) provides a detectable signal; (ii) interacts    with a second label to modify the detectable signal provided by the    first or second label; or (iii) confers a capture function, i.e.    hydrophobic affinity, antibody/antigen, ionic complexation.-   h. As used herein, “sequence specifically” means hybridization by    base pairing through hydrogen bonding. Non-limiting examples of    standard base pairing includes adenine base pairing with thymine or    uracil and guanine base pairing with cytosine. Other non-limiting    examples of base-pairing motifs include, but are not limited to:    adenine base pairing with any of: 5-propynyl-uracil,    2-thio-5-propynyl-uracil, 2-thiouracil or 2-thiothymine; guanine    base pairing with any of: 5-methylcytosine or pseudoisocytosine;    cytosine base pairing with any of: hypoxanthine,    N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine); thymine or    uracil base pairing with any of: 2-aminopurine,    N9-(2-amino-6-chloropurine) or N9-(2,6-diaminopurine); and    N8-(7-deaza-8-aza-adenine), being a universal base, base pairing    with any other nucleobase, such as for example any of: adenine,    cytosine, guanine, thymine, uracil, 5-propynyl-uracil,    2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,    2-thiouracil and 2-thiothymine, 2-aminopurine,    N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,    N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine) (See: Seela et    al., Nucl. Acids, Res.: 28(17): 3224-3232 (2000)).-   i. As used herein, the term “chimera” or “chimeric oligomer” means    an oligomer comprising two or more linked subunits that are selected    from different classes of subunits. For example, a PNA/DNA chimera    would comprise at least two PNA subunits linked to at least one    2′-deoxyribonucleic acid subunit (For exemplary methods and    compositions related to PNA/DNA chimera preparation See:    WO96/40709). Exemplary component subunits of the chimera are    selected from the group consisting of PNA subunits, naturally    occurring amino acid subunits, DNA subunits, RNA subunits and    subunits of analogues or mimics of nucleic acids.-   j. As used herein, the term “linked polymer” means a polymer    comprising two or more polymer segments which are linked by a    linker. The polymer segments that are linked to form the linked    polymer are selected from the group consisting of an    oligodeoxynucleotide, an oligoribonucleotide, a peptide, a    polyimide, a peptide nucleic acid (PNA) and a chimera.-   k. As used herein “solid support” or “solid carrier” means any solid    phase material upon which a oligomer is synthesized, attached,    ligated or otherwise immobilized. Solid support encompasses terms    such as “resin”, “solid phase”, “surface” and “support”. A solid    support may be composed of organic polymers such as polystyrene,    polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy,    and polyacrylamide, as well as co-polymers and grafts thereof. A    solid support may also be inorganic, such as glass, silica,    controlled-pore-glass (CPG), or reverse-phase silica. The    configuration of a solid support may be in the form of beads,    spheres, particles, granules, a get, or a surface. Surfaces may be    planar, substantially planar, or non-planar. Solid supports may be    porous or non-porous, and may have swelling, or non-swelling    characteristics. A solid support may be configured in the form of a    well, depression or other container, vessel, feature or location. A    plurality of solid supports may be configured in an array at various    locations, addressable for robotic delivery of reagents, or by    detection means including scanning by laser illumination and    confocal or deflective light gathering.-   l. As used herein, “support bound” means immobilized on or to a    solid support. It is understood that immobilization can occur by any    means, including for example; by covalent attachment, by    electrostatic immobilization, by attachment through a ligand/ligand    interaction, by contact or by depositing on the surface.    2. Description    I. General:    PNA Synthesis:

Methods for the chemical assembly of PNAs are well known (See: U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,5,986,053 and 6,107,470; all of which are herein incorporated byreference (Also see: PerSeptive Biosystems Product Literature)). As ageneral reference for PNA synthesis methodology also please see: Nielsenet al., Peptide Nucleic Acids; Protocols and Applications, HorizonScientific Press, Norfolk England (1999).

Chemicals and instrumentation for the support bound automated chemicalassembly of peptide nucleic acids are now commercially available. Bothlabeled and unlabeled PNA oligomers are likewise available fromcommercial vendors of custom PNA oligomers. Chemical assembly of a PNAis analogous to solid phase peptide synthesis, wherein at each cycle ofassembly the oligomer possesses a reactive alkyl amino terminus that canbe condensed with the next synthon to be added to the growing polymer.Because standard peptide chemistry is utilized, natural and non-naturalamino acids can be routinely incorporated into a PNA oligomer. Because aPNA is a polyamide, it has a C-terminus (carboxyl terminus) and anN-terminus (amino terminus). For the purposes of the design of ahybridization probe suitable for antiparallel binding to the targetsequence (the preferred orientation), the N-terminus of the probingnucleobase sequence of the PNA probe is the equivalent of the5′-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.

PNA Labeling:

Non-limiting methods for labeling PNAs are described in U.S. Pat. No.6,110,676, U.S. Pat. No. 6,361,942, U.S. Pat. No. 6,355,421 (allincorporated herein by reference), WO99/21881, the examples section ofthis specification or are otherwise well known in the art of PNAsynthesis. Other non-limiting examples for labeling PNAs are alsodiscussed in Nielsen et al., Peptide Nucleic Acids; Protocols andApplications, Horizon Scientific Press, Norfolk England (1999).

Labels:

Non-limiting examples of detectable moieties (labels) that can be usedto label PNA probes or antibodies used in the practice of this inventioncan include a dextran conjugate, a branched nucleic acid detectionsystem, a chromophore, a fluorophore, a spin label, a radioisotope, anenzyme, a hapten, an acridinium ester or a chemiluminescent compound.Other suitable labeling reagents and preferred methods of attachmentwould be recognized by those of ordinary skill in the art of PNA,peptide or nucleic acid synthesis.

Non-limiting examples of haptens include 5(6)-carboxytluorescein,2,4-dinitrophenyl, digoxigenin, and biotin.

Non-limiting examples of fluorochromes (fluorophores) include5(6)-carboxyfluorescein (Flu),6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid (Cou), 5(and6)-carboxy-X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye,Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) DyeCyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3, 3.5, 5 and5.5 are available as NHS esters from Amersham, Arlington Heights, Ill.)or the Alexa dye series (Molecular Probes, Eugene, Oreg.).

Non-limiting examples of enzymes include polymerases (e.g. Taqpolymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNApolymerase 1 and phi29 polymerase), alkaline phosphatase (AP),horseradish peroxidase (HRP), soy bean peroxidase (SBP)), ribonucleaseand protease.

Energy Transfer

In one embodiment, PNA oligomers can be labeled with an energy transferset. For energy transfer to be useful in determining hybridization,there should be an energy transfer set comprising at least one energytransfer donor and at least one energy transfer acceptor moiety. Often,the energy transfer set will include a single donor moiety and a singleacceptor moiety, but this is not a limitation. An energy transfer setmay contain more than one donor moiety and/or more than one acceptormoiety. The donor and acceptor moieties operate such that one or moreacceptor moieties accept energy transferred from the one or more donormoieties or otherwise quench the signal from the donor moiety ormoieties. Thus, in one embodiment, both the donor moiety(ies) andacceptor moiety(ies) are fluorophores. Though the previously listedfluorophores (with suitable spectral properties) might also operate asenergy transfer acceptors, the acceptor moiety can also be anon-fluorescent quencher moiety such as4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl). The labels ofthe energy transfer set can be linked at the oligomer termini or linkedat a site within the oligomer. For example, each of two labels of anenergy transfer set can be linked at the distal-most termini of theoligomer.

Transfer of energy between donor and acceptor moieties may occur throughany energy transfer process, such as through the collision of theclosely associated moieties of an energy transfer set(s) or through anon-radiative process such as fluorescence resonance energy transfer(FRET). For FRET to occur, transfer of energy between donor and acceptormoieties of a energy transfer set requires that the moieties be close inspace and that the emission spectrum of a donor(s) have substantialoverlap with the absorption spectrum of the acceptor(s) (See: Yaron etal. Analytical Biochemistry, 95: 228-235 (1979) and particularly page232, col. 1 through page 234, col. 1). Alternatively, collision mediated(radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies) (See: Yaron et al.,Analytical Biochemistry, 95: 228-235 (1979) and particularly page 229,col. 1 through page 232, col. 1). This process is referred to asintramolecular collision since it is believed that quenching is causedby the direct contact of the donor and acceptor moieties (See: Yaron etal.). It is to be understood that any reference to energy transfer inthe instant application encompasses all of these mechanisticallydistinct phenomena. It is also to be understood that energy transfer canoccur though more than one energy transfer process simultaneously andthat the change in detectable signal can be a measure of the activity oftwo or more energy transfer processes. Accordingly, the mechanism ofenergy transfer is not a limitation of this invention.

Detecting Energy Transfer in a Self-Indicating PNA Oligomer:

When labeled with an energy transfer set, we refer to the PNA oligomeras being self-indicating. In one embodiment, a self-indicating PNAoligomer can be labeled in a manner that is described in co-pending andcommonly owned patent application U.S. Ser. No. 09/179,162 (nowallowed), entitled: “Methods, Kits And Compositions Pertaining To LinearBeacons” and the related PCT application which has also now published asWO99/21881, both of which are hereby incorporated by reference.

Hybrid formation between a self-indicating oligomer and a targetsequence can be monitored by measuring at least one physical property ofat least one member of the energy transfer set that is detectablydifferent when the hybridization complex is formed as compared with whenthe oligomer exists in a non-hybridized state. We refer to thisphenomenon as the self-indicating property of the oligomer. This changein detectable signal results from the change in efficiency of energytransfer between donor and acceptor moieties caused by hybridization ofthe oligomer to the target sequence.

For example, the means of detection can involve measuring fluorescenceof a donor or acceptor fluorophore of an energy transfer set. In oneembodiment, the energy transfer set may comprise at least one donorfluorophore and at least one acceptor (fluorescent or non-fluorescent)quencher such that the measure of fluorescence of the donor fluorophorecan be used to detect, identify or quantitate hybridization of theoligomer to the target sequence. For example, there may be a measurableincrease in fluorescence of the donor fluorophore upon the hybridizationof the oligomer to a target sequence.

In another embodiment, the energy transfer set comprises at least onedonor fluorophore and at least one acceptor fluorophore such that themeasure of fluorescence of either, or both, of at least one donor moietyor one acceptor moiety can be used to can be used to detect, identify orquantitate hybridization of the oligomer to the target sequence.

Self-indicating PNA oligomers can be used in in-situ hybridizationassays. However, certain self-indicating PNA oligomers are particularlywell suited for the analysis of nucleic acid amplification reactions(e.g. PCR) either in real-time or at the end point (See: WO99/21881).

Determining Energy Transfer in a Detection Complex:

In another embodiment, the PNA oligomers of the present invention arelabeled solely with a quencher moiety and can be used as a componentoligomer in a Detection Complex as more fully explained in U.S. Pat. No.6,361,942, entitled: “Methods, Kits And Compositions Pertaining ToDetection Complexes”, herein incorporated by reference. When theDetection Complex is formed, at least one donor moiety of one componentpolymer is brought sufficiently close in space to at least one acceptormoiety of a second component polymer. Since the donor and acceptormoieties of the set are closely situated in space, transfer of energyoccurs between moieties of the energy transfer set. When the DetectionComplex dissociates, as for example when one of the component polymersof the Detection Complex hybridizes to a target sequence, the donor andacceptor moieties do not interact sufficiently to cause substantialtransfer of energy from the donor and acceptor moieties of the energytransfer set and there is a correlating change in detectable signal fromthe donor and/or acceptor moieties of the energy transfer set.Consequently, Detection Complex formation/dissociation can be determinedby measuring at least one physical property of at least one member ofthe energy transfer set that is detectably different when the complex isformed as compared with when the component polymers of the DetectionComplex exist independently and unassociated.

Detectable and Independently Detectable Moieties/Multiplex Analysis:

A multiplex hybridization assay can be performed in accordance with thisinvention. In a multiplex assay, numerous conditions of interest can besimultaneously examined. Multiplex analysis relies on the ability tosort sample components or the data associated therewith, during or afterthe assay is completed. In preferred embodiments of the invention, oneor more distinct independently detectable moieties can be used to labeltwo or more different probes used in an assay. The ability todifferentiate between and/or quantitate each of the independentlydetectable moieties provides the means to multiplex a hybridizationassay because the data that correlates with the hybridization of each ofthe distinctly (independently) labeled probe to a particular nucleicacid sequence can be correlated with the presence, absence or quantityof each organism sought to be detected in the sample. Consequently, themultiplex assays of this invention can be used to simultaneously detectthe presence, absence or quantity of two or more different organisms(e.g. species of Candida) in the same sample and in the same assay. Forexample, a multiplex assay may utilize two or more PNA probes, eachbeing labeled with an independently detectable fluorophore, or a set ofindependently detectable fluorophores.

Spacer/Linker Moieties:

Generally, spacers are used to minimize the adverse effects that bulkylabeling reagents might have on hybridization properties of probes.Linkers typically induce flexibility and randomness into the probe orotherwise link two or more nucleobase sequences of a probe or componentpolymer. Preferred spacer/linker moieties for the nucleobase polymers ofthis invention consist of one or more aminoalkyl carboxylic acids (e.g.aminocaproic acid) the side chain of an amino acid (e.g. the side chainof lysine or ornithine), natural amino acids (e.g. glycine),arninooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e.g. succinic acid), alkyloxy diacids (e.g. diglycolic acid) oralkyldiamines (e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moietiesmay also incidentally or intentionally be constructed to improve thewater solubility of the probe (For example see: Gildea et al., Tett.Lett. 39: 7255-7258 (1998)).

For example, a spacer/linker moiety can comprise one or more linkedcompounds having the formula: —Y—(O_(m)—(CW₂)_(n))_(o)—Z—. The group Yis selected from the group consisting of: a single bond, —(CW₂)_(p)—,—C(O)(CW)_(p)—, —C(S)(CW₂)_(p)— and —S(O₂)(CW₂)_(p). The group Z has theformula NH, NR², S or O. Each W is independently H, R², —OR², F, Cl, Bror I; wherein, each R² is independently selected from the groupconsisting of: —CX₃, —CX₂CX₃, —CX₂CX₂CX₃, —CX₂CX(CX₃)₂, and —C(CX₃)₃.Each X is independently H, F, Cl, Br or I. Each m is independently 0or 1. Each n, o and p are independently integers from 0 to 10.

Hybridization. Conditions/Stringency:

Those of ordinary skill in the art of nucleic acid hybridization willrecognize that factors commonly used to impose or control stringency ofhybridization include formamide concentration (or other chemicaldenaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probe/targetcombination can often be found by the well known technique of fixingseveral of the aforementioned stringency factors and then determiningthe effect of varying a single stringency factor. The same stringencyfactors can be modulated to thereby control the stringency ofhybridization of a PNA to a nucleic acid, except that the hybridizationof a PNA is fairly independent of ionic strength. Optimal stringency foran assay may be experimentally determined by examination of eachstringency factor until the desired degree of discrimination isachieved.

Suitable Hybridization Conditions:

Generally, the more closely related the background causing nucleic acidcontaminates are to the target sequence, the more careful stringencymust be controlled. Blocking probes may also be used as a means toimprove discrimination beyond the limits possible by mere optimizationof stringency factors. Suitable hybridization conditions will thuscomprise conditions under which the desired degree of discrimination isachieved such that an assay generates an accurate (within the tolerancedesired for the assay) and reproducible result. Aided by no more thanroutine experimentation and the disclosure provided herein, those ofskill in the art will easily be able to determine suitable hybridizationconditions for performing assays utilizing the methods, kits andcompositions described herein. Suitable in-situ hybridization conditionscomprise conditions suitable for performing an in-situ hybridizationprocedure. Thus, suitable hybridization or suitable in-situhybridization conditions will become apparent using the disclosureprovided herein; with or without additional routine experimentation.

Blocking Probes:

Blocking probes are nucleic acid or non-nucleic acid probes (e.g. PNAprobes) that can be used to suppress the binding of the probingnucleobase sequence of the probing polymer to a non-target sequence.Preferred blocking probes are PNA probes (See: Coull et al., WIPOpublication No. WO98/24933 as well as U.S. Pat. No. 6,110,676).Typically, blocking probes are closely related to the probing nucleobasesequence and preferably they comprise a point mutation as compared withthe probing nucleobase sequence. It is believed that blocking probesoperate by hybridization to the non-target sequence to thereby form amore thermodynamically stable complex than is formed by hybridizationbetween the probing nucleobase sequence and the non-target sequence.Formation of the more stable and preferred complex blocks formation ofthe less stable non-preferred complex between the probing nucleobasesequence and the non-target sequence. Thus, blocking probes can be usedwith the methods, kits and compositions of this invention to suppressthe binding of the PNA probe to a non-target sequence that might bepresent and interfere with the performance of the assay. Blocking probesare particularly advantageous in single point mutation discrimination.

Probing Nucleobase Sequence:

The probing nucleobase sequence of a PNA probe is the specific sequencerecognition portion of the construct. Therefore, the probing nucleobasesequence is a sequence of PNA subunits designed to sequence specificallyhybridize to a target sequence wherein the presence, absence and/oramount of target sequence can be used to detect the presence, absenceand/or number of organisms of interest in a sample. Consequently, withdue consideration of the requirements of a PNA probe for the assayformat chosen, the length of the probing nucleobase sequence of the PNAprobe will generally be chosen such that a stable complex is formed withthe target sequence under suitable hybridization conditions or suitablein-situ hybridization conditions.

The probing nucleobase sequence suitable for detecting the targetorganism listed in Table 1, will generally, but not necessarily, have alength of 18 or fewer PNA subunits wherein the exact nucleobase sequencecan be at least 90% homologous to the probing nucleobase sequenceslisted in Table 1, or their complements. The PNA probes can be 100%homologous to said sequences or can comprise the exact nucleobasesequences appearing the Table 1. Complements of the probing nucleobasesequence are included since it is possible to prepare or amplify copiesof the target sequence wherein the copies are complements of the targetsequence and thus, will bind to the complement of the probing nucleobasesequences listed in Table 1. Useful probing nucleobase sequences arelisted in Table 1. These probing nucleobase sequences have been shown tobe relatively or highly specific for the target organism indicated inthe presence of other organisms, including the other species of Candida(See information listed in Table 1 and the Examples, below).

A PNA probe of this invention will generally have a probing nucleobasesequence that is complementary to the target sequence. Alternatively, asubstantially complementary probing nucleobase sequence might be usedsince it has been demonstrated that greater sequence discrimination canbe obtained when utilizing probes wherein there exists one or more pointmutations (base mismatch) between the probe and the target sequence(See: Guo et al., Nature Biotechnology 75:331-335 (1997)).

This invention contemplates that variations in the probing nucleobasesequences listed in Table 1 shall provide PNA probes that are suitablefor the specific detection of the organisms listed. Common variationsinclude, deletions, insertions and frame shifts. Variation of theprobing nucleobase sequences within the parameters described herein areconsidered to be an embodiment of this invention.

Probe Complexes:

In still another embodiment, two probes are designed to hybridize to thetarget sequence sought to be detected to thereby generate a detectablesignal whereby the probing nucleobase sequence of each probe compriseshalf or approximately half of the nucleobase sequence required forhybridization to the complete target sequence of the organism sought tobe detected in the assay such that the aggregate nucleobase sequence ofthe two probes forms the probing nucleobase sequence that hybridizes tothe target sequence. As a non-limiting example, the probing nucleobasesequences of the two probes might be designed using the assay asdescribed in U.S. Pat. No. 6,027,893, entitled: “Method of identifying anucleic acid using triple helix formation of adjacently annealed probes”by H. Orum et al., herein incorporated by reference. Using thismethodology, the probes that hybridize to the target sequence may or maynot be labeled. However, it is the probe complex formed by the annealingof the adjacent probes that is detected. Similar compositions comprisedsolely of PNA probes have been described in U.S. Pat. No. 6,287,772,herein incorporated by reference.

II. Preferred Embodiments of the Invention:

a. PNA Probes:

In one embodiment, this invention is directed to PNA probes. The PNAprobes of this invention are suitable for detecting, identifying and/orquantitating one or more species of Candida in a sample. The PNA probes,probe sets, methods and kits of this invention are suitable for theanalysis of nucleic acid, whether or not it is present within anorganism of interest. Accordingly, this invention can be used for boththe analysis of organisms or for the analysis of nucleic acid extractedfrom or derived from an organism of interest.

With the exception of Seq. Id. Nos. 9 and 10 (See: Table 1 for a list oftarget organisms), generally the PNA probes comprising the identifiedprobing nucleobase sequences that are specific for a certain species ofCandida. General characteristics (e.g. length, labels, linkers etc.) ofPNA probes suitable for the detection, identification or quantitation ofthese specific organisms have been previously described herein.Non-limiting examples of probing nucleobase sequences of PNA probes ofthis invention are listed in Table 1, below. The species of Candida thatthe probing nucleobase sequence is designed to determine has also beenidentified in Table 1 as the target organism.

The PNA probes of this invention may comprise only a probing nucleobasesequence (as previously described herein) or may comprise additionalmoieties. Non-limiting examples of additional moieties includedetectable moieties (labels), linkers, spacers, natural or non-naturalamino acids, peptides, enzymes and/or other subunits of PNA, DNA or RNA.Additional moieties may be functional or non-functional in an assay.Generally however, additional moieties will be selected to be functionalwithin the design of the assay in which the PNA probe is to be used. Forexample, the PNA probes of this invention can be labeled with one ormore detectable moieties or labeled with two or more independentlydetectable moieties. The independently detectable moieties can beindependently detectable fluorophores.

TABLE 1 Seq. ID. Target No. Organism Probing Nucleobase Sequence  1C. albicans AGA-GAG-CAG-CAT-GCA  2 C. albicans AGA-GAG-CAA-CAT-GCA  3C. albicans ACA-GCA-GAA-GCC-GTG  4 C. albicans CAT-AAA-TGG-CTA-CCA-GA  5C. albicans CAT-AAA-TGG-CTA-CCC-AG  6 C. albicans ACT-TGG-AGT-CGA-TAG  7C. albicans CCA-AGG-CTT-ATA-CTC-GC  8 C. albicans CCC-CTG-AAT-CGG-GAT  9C. albicans & GAC-GCC-AAA-GAC-GCC C. dubliniesis 10 C. albicans &ATC-GTC-AGA-GGC-TAT-AA C. dubliniesis 11 C. dubliniesisTAG-CCA-GAA-GAA-AGG 12 C. dubliniesis CAT-AAA-TGG-CTA-GCC-AG 13C. dubliniesis CTC-CGA-TGT-GAC-TGC-G 14 C. dubliniesisTCC-CAG-ACT-GCT-CGG 15 C. glabrata TCC-AAG-AGG-TCG-AGA 16 C. glabrataGCC-AAG-CCA-CAA-GGA 17 C. glabrata GCC-GCC-AAG-CCA-CA 18 C. glabrataGGA-CTT-GGG-GTT-AG 19 C. glabrata CCG-GGT-GCA-TTC-CA 20 C. kruseiATG-TAG-AAC-GGA-ACT-A 21 C. krusei GAT-TCT-CGG-CCC-CAT-G 22C. parapsilosis CTG-GTT-CGC-CAA-AAA-G 23 C. tropicalisAGT-ACG-CAT-CAG-AAA

The probes of this invention can be used in in-situ hybridization (ISH)and fluorescence in-situ hybridization (FISH) assays. Excess probe usedin an ISH or FISH assay often will be removed so that the detectablemoiety of specifically bound probes can be detected above the backgroundsignal that results from still present but unhybridized probe.Generally, the excess probe can be washed away after the sample has beenincubated with probe for a period of time. However, because certaintypes of self-indicating probes can generate little or no detectablebackground, they can be used to eliminate the requirement that excessprobe be completely removed (washed away) from the sample.

Unlabeled Non-Nucleic Acid Probes:

The probes of this invention need not be labeled with a detectablemoiety to be operable within the scope of this invention. When using theprobes of this invention it is possible to detect the probe/targetsequence complex formed by hybridization of the probing nucleobasesequence of the probe to the target sequence. For example, a PNA/nucleicacid complex formed by the hybridization of a PNA probing nucleobasesequence to the target sequence could be detected using an antibody thatspecifically interacts with the complex under antibody bindingconditions. Suitable antibodies to PNA/nucleic acid complexes andmethods for their preparation and use are described in WIPO PatentApplication WO95/17430 and U.S. Pat. No. 5,612,458, herein incorporatedby reference.

The antibody/PNA/nucleic acid complex formed by interaction of theα-PNA/nucleic acid antibody with the PNA/nucleic acid complex can bedetected by several methods. For example, the α-PNA/nucleic acidantibody could be labeled with a detectable moiety. Suitable detectablemoieties have been previously described herein. Thus, the presence,absence and/or quantity of the detectable moiety can be correlated withthe presence, absence and/or quantity of the antibody/PNA/nucleic acidcomplex and the species of Candida to be identified by the probingnucleobase sequence of the PNA probe. Alternatively, theantibody/PNA/nucleic acid complex can be detected using a secondaryantibody that is labeled with a detectable moiety. Typically thesecondary antibody specifically binds to the α-PNA/nucleic acid antibodyunder antibody binding conditions. Thus, the presence, absence and/orquantity of the detectable moiety can be correlated with the presence,absence and/or quantity of the antibody/antibody/PNA/nucleic acidcomplex and the species of Candida to be identified by the probingnucleobase sequence of the probe. As used herein, the term antibodyincludes antibody fragments that specifically bind to other antibodiesor other antibody fragments.

Immobilization of Probes to a Surface:

One or more of the PNA probes of this invention may optionally beimmobilized to a surface for the detection of the target sequence of atarget organism of interest. PNA probes can be immobilized to thesurface using the well known process of UV-crosslinking. A PNA probe canbe synthesized on the surface in a manner suitable for deprotection butnot cleavage from the synthesis support (See: Weiler, J. et al,Hybridization based DNA screening on peptide nucleic acid (PNA) oligomerarrays, Nucl. Acids Res., 25, 24:2792-2799 (July 1997)). In stillanother embodiment, PNA probes can be covalently linked to a surface bythe reaction of a suitable functional group on the probe with afunctional group of the surface (See: Lester, A. et al, “PNA ArrayTechnology”: Presented at Biochip Technologies Conference in Annapolis(October 1997)). This method is most advantageous since the PNA probeson the surface will typically be highly purified and attached using adefined chemistry, thereby minimizing or eliminating non-specificinteractions.

Methods for the chemical attachment of probes to surfaces generallyinvolve the reaction of a nucleophilic group, (e.g. an amine or thiol)of the probe to be immobilized, with an electrophilic group on thesupport to be modified. Alternatively, the nucleophile can be present onthe support and the electrophile (e.g. activated carboxylic acid)present on the probe. Because native PNA possesses an amino terminus, aPNA will not necessarily require modification to thereby immobilize itto a surface (See: Lester et al., Poster entitled “PNA ArrayTechnology”).

Conditions suitable for the immobilization of a PNA probe to a surfacewill generally be similar to those conditions suitable for the labelingof the polymer. The immobilization reaction is essentially theequivalent of labeling whereby the label is substituted with the surfaceto which the polymer is to be linked.

Numerous types of surfaces derivatized with amino groups, carboxylicacid groups, isocyantes, isothiocyanates and malimide groups arecommercially available. Non-limiting examples of suitable surfacesinclude membranes, chips (e.g. silicone chips), glass, controlled poreglass, polystyrene particles (beads), silica and gold nanoparticles.

Arrays of PNA Probes or Probe Sets:

Arrays are surfaces to which two or more probes have been immobilizedeach at a specified position. The probing nucleobase sequence of theimmobilized probes can be judiciously chosen to interrogate a samplethat may contain nucleic acid from one or more target organisms. Becausethe location and composition of each immobilized probe is known, arrayscan be useful for the simultaneous detection, identification and/orquantitation of nucleic acid from two or more target organisms that maybe present in the sample. Moreover, arrays of PNA probes can beregenerated by stripping away any of the hybridized nucleic acid aftereach assay, thereby providing a means to repetitively analyze numeroussamples using the same array. Thus, arrays of PNA probes or PNA probesets may be useful for repetitive screening of samples for targetorganisms of interest. The arrays of this invention comprise at leastone PNA probe (as described herein) suitable for the detection,identification and/or quantitation of at least one species of Candida.Exemplary probing nucleobase sequences for the immobilized PNA probesare listed in Table 1.

b. PNA Probe Sets:

In another embodiment, this invention is directed to a PNA probe setsuitable for detecting, identifying and/or quantitating one or morespecies of Candida yeast in a sample of interest wherein at least one ofthe species sought to be detected with the probe set is selected fromthe group consisting of: C. albicans, C. dubliniensis, C. krusei, C.glabrata, C. parapsilosis and C. tropicalis. The general and preferredcharacteristics of PNA probes suitable for the detection, identificationand/or quantitation of these specific yeast species have been previouslydescribed herein. Preferred probing nucleobase sequences for the targetspecies are listed in Table 1. The grouping of PNA probes within setscharacterized for specific groups of species can be a very usefulembodiment of this invention. The PNA probes of this invention can becombined with probes for other yeast or even for organisms other thanyeast such as been described in U.S. Pat. No. 6,280,946, hereinincorporated by reference, wherein a multiplex assay for both yeast andbacteria has been described using a PNA probe set.

Probe sets of this invention comprise at least one PNA probe but neednot comprise only PNA probes. For example, probe sets of this inventionmay comprise mixtures of PNA probes and nucleic acid probes, providedhowever that a set comprises at least one PNA probe as described herein.In one embodiment, some of the probes of the set can be blocking probescomposed of PNA or nucleic acid. In other embodiments, the probe set canbe used to determine organisms other than species of Candida in additionto the determination of at least one species of Candida.

Table 1 lists several species of Candida for which two or more probingnucleobase sequences are identified as being suitable for detecting theidentified target organism. Where alternative probing nucleobasesequences exist, it can be advantageous to use a probe set containingthe two or more PNA probes to thereby increase the detectable signal inthe assay.

One exemplary probe set would comprise probes suitable for determiningC. albicans wherein two or more of the probes of the set comprise aprobing nucleobase sequence selected from the group consisting of:AGA-GAG-CAG-CAT-GCA (Seq. Id. No. 1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No.2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3), CAT-AAA-TGG-CTA-CCA-GA (Seq.Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5), ACT-TGG-AGT-CGA-TAG(Seq. Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7) andCCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8). A second exemplary probe set cancomprise probes suitable for determining C. albicans and C. dubliniensiswherein the probes of the set comprise a probing nucleobase sequenceselected from the group consisting of: GAC-GCC-AAA-GAC-GCC (Seq. Id. No.9) and ATC-GTC-AGA-GGC-TAT-AA (Seq. Id. No. 10). Still a third exemplaryprobe set can comprise probes suitable for determining only C.dubliniensis wherein at least two the probes of the set comprise aprobing nucleobase sequence selected from the group consisting of:TAG-CCA-GAA-GAA-AGG (Seq. Id. No. 11), CAT-AAA-TGG-CTA-GCC-AG (Seq. Id.No. 12), CTC-CGA-TGT-GAC-TGC-G (Seq. Id. No. 13) and TCC-CAG-ACT-GCT-CGG(Seq. Id. No. 14). Yet a fourth exemplary probe set can comprise probessuitable for determining only C. glabrata wherein at least two theprobes of the set comprise a probing nucleobase sequence selected fromthe group consisting of: TCC-AAG-AGG-TCG-AGA (Seq. Id. No. 15),GCC-AAG-CCA-CAA-GGA (Seq. Id. No. 16), GCC-GCC-AAG-CCA-CA (Seq. Id. No.17), GGA-CTT-GGG-GTT-AG (Seq. Id. No. 18) and CCG-GGT-GCA-TTC-CA (Seq.Id. No. 19) Still a fifth exemplary probe set can comprise probeswherein the probing nucleobase sequence is selected from the groupconsisting of: ATG-TAG-AAC-GGA-ACT-A (Seq. Id. No. 20) andGAT-TCT-CGG-CCC-CAT-G (Seq. Id. No. 21).

In other embodiments, the probe set can comprise two or more probes suchthat two or more of the species of Candida identified herein aredetected, identified and/or quantitated. Preferably, the set comprisestwo or more independently detectable PNA probes wherein eachindependently detectable probe is suitable for detecting, identifyingand/or quantitating a particular species of Candida. For example, a PNAprobe set comprising Seq. Id. Nos. 1 and 11, wherein each probe islabeled with an independently detectable moiety and can be used in twoindependent assays, or a single multiplex assay, for the independentdetermination of both C. albicans and C. dubliniensis (See: Example 2for a non-multiplex assay demonstrates that these species can beindependently determined using the probing nucleobase sequencesidentified herein).

c. Methods:

In another embodiment, this invention is directed to a method suitablefor detecting, identifying and/or quantitating one or more species ofCandida in a sample of interest. According to the method, one or moreindividual species of Candida can be determined. The general andpreferred characteristics of PNA probes suitable for the detection,identification and/or quantitation of these target organisms have beenpreviously described herein. Exemplary probing nucleobase sequences arelisted in Table 1.

In one embodiment, the method can comprise contacting the sample withone or more PNA probes, wherein suitable probes have been previouslydescribed herein. According to the method, the presence, absence and/ornumber of the one or more species of Candida in the sample can bedetected, identified and/or quantitated by correlating hybridization ofthe probing nucleobase sequence of one or more PNA probes to the targetsequence of a target organism of interest under suitable hybridizationconditions or suitable in-situ hybridization conditions. The grouping ofPNA probes within probe sets to be used with methods for detectingspecific organisms or groups of organisms can also be done. Exemplaryprobes and probe sets suitable for the practice of this method have beenpreviously described herein. Preferred methods for the determination ofyeast, with or without the simultaneous detection of bacteria, have beenpreviously described in U.S. Pat. No. 6,280,946, incorporated herein byreference. Examples 1 and 2, below, provide further methods for thedetermination of the specific yeast identified herein.

Exemplary Assay Formats:

The probes, probe sets, methods and kits of this invention can be usedfor the detection, identification and/or quantitation of Candida yeast.In-situ hybridization (ISH) or fluorescent in-situ hybridization (FISH)can be used as the assay format: for detecting, identifying and/orquantitating target organisms. Specific PNA-RSH methods used toexperimentally test specific PNA probes can be found in Examples 1 and 2of this specification. The examples contained herein demonstrate thatlabeled PNA probes comprising the probing nucleobase sequences listed inTable 1 are specific for determining target organisms even when otherorganisms listed in the Table are present in the assay. The experimentalconditions used in the Examples yield results within approximately 1-4hours. The identical experimental protocol was found to be specific,reliable and generally applicable regardless of the nature or sequenceof the PNA probes used.

Organisms that have been treated with the PNA probes or probe sets orkits described herein can be determined by several exemplary methods.The cells can be fixed on slides and visualized with a film, camera,slide scanner or microscope. Alternatively, the cells can be fixed andthen analyzed in a flow cytometer. Slide scanners and flow cytometersare particularly useful for rapidly quantitating the number of targetorganisms present in a sample of interest.

d. Kits:

In yet another embodiment, this invention is directed to kits suitablefor performing an assay that detects the presence, absence and/orquantity of Candida yeast in a sample. The general and preferredcharacteristics of PNA probes suitable for the detection, identificationand/or quantitation of Candida have been previously described herein.Exemplary probing nucleobase sequences are listed in Table 1.Furthermore, methods suitable for using the PNA probes or PNA probe setsof a kit suitable to detect, identify and/or quantitate target organismsin a sample of interest have been previously described herein.

The kits of this invention comprise one or more PNA probes and otherreagents or compositions that are selected to perform an assay orotherwise simplify the performance of an assay. The kits can, forexample, comprise buffers and/or other reagents useful for performing aPNA-ISH or PNA-FISH assay. In other embodiments, the buffers and/orother reagents can be useful for performing a nucleic acid amplificationreaction such as a PCR reaction.

In kits that contain sets of probes, wherein each of at least two probesof the set are used to detect different species of Candida, the probesof the set can be labeled with one or more independently detectablemoieties so that each specific target organism can be individuallydetected, identified and/or quantitated in a single assay (e.g. amultiplex assay).

e. Exemplary Applications for Using the Invention:

Whether support bound or in solution, the PNA probes, probe sets,methods and kits of this invention can be useful for the rapid,sensitive and reliable detection of Candida yeast in food, beverages,water, pharmaceutical products, personal care products, dairy productsor for the analysis of environmental samples. The analysis of beveragescan include soda, bottled water, fruit juice, beer, wine or liquorproducts. Suitable PNA probes, probe sets, methods and kits of thisinvention can be particularly useful for the analysis of raw materials,equipment, products or processes used to manufacture or store food,beverages, water, pharmaceutical products, personal care products, dairyproducts or for the analysis of environmental samples.

Whether support bound or in solution; the PNA probes, probe sets,methods and kits of this invention are can be useful for the detectionof Candida yeast in clinical samples and clinical environments. By wayof a non-limiting example, the PNA probes, probe sets, methods and kitsof this invention can be particularly useful in the analysis of bloodculture samples. Other non-limiting examples of clinical samplesinclude: sputum, laryngeal swabs, gastric lavage, bronchial washings,biopsies, aspirates, expectorates, body fluids (e.g. spinal, pleural,pericardial, synovial, blood, pus, amniotic, and urine), bone marrow andtissue sections. Suitable PNA probes, probe sets, methods and kits canalso be particularly useful for the analysis of clinical specimens,equipment, fixtures or products used to treat humans or animals.

EXAMPLES

This invention is now illustrated by the following examples that are notintended to be limiting in any way.

All PNA oligomers were prepared using conventional synthesis andpurification procedures.

Example 1 Analysis of a PNA Probe Specific to Candida albicans

PNA Probe sequence Can26S03/Flu Flu-O-AGAGAGCAGCATGCA-NH₂ Note: Flu =5(6)-carboxy-fluorescein; O = 8-amino-3,6-dioxaoctanoic acid

Reference Strains and Clinical Isolates. Fourteen C. albicans referencestrains and nineteen other reference strains representingphylogenetically related Candida species and Lodderomyces elongisporus,mainly within the C. albicans clade, were selected from the AgriculturalResearch Service Culture Collection (NRRL) Peoria, Ill. OneSaccharomyces cerevisiae strain was obtained from the American TypeCulture Collection (ATCC), Manassas, Va. Fifty-eight C. dubliniensis andthirty-nine C. albicans clinical isolates were collected, at theInstitute of Medical Microbiology, University Hospital, Aachen, Germany.The C. dubliniensis isolates were mainly from HIV-positive patients andfrom respiratory specimens of patients with cystic fibrosis. Clinicalisolates of C. albicans were from various clinical specimens, includingblood cultures, and chosen to represent different strains, i.e. serotypeA, the biovar stellatoidea as well as phenotypically aberrant isolatessuch as red pigment strain and strains that failed to assimilateglucosamine and N-acetylglucosamine. All strains and isolates wereidentified by D1/D2 26S rDNA sequence analysis. For PNA FISH analysis,reference strains and clinical isolates were inoculated into yeast-malt(YM) broth (Difco Laboratories, Detroit, Mich.) and incubated overnightat 35° C. Furthermore, thirty-three C. albicans isolates and eighteenother isolates representing clinically significant yeast speciesobtained from various clinical specimens, including blood cultures(Clinical Microbiology Laboratory, Cleveland Clinic Foundation, OH),were spiked into FAN BacT/Alert medium (Organon Teknika, Durham, N.C.)and incubated in the BacT/Alert Microbial detection system (OrganonTeknika) until they were detected as positive by the system. Theeighteen non-C. albicans isolates comprised C. glabrata (n=5), C.tropicalis (n=3), C. krusei (n=2), C. parapsilosis (n=4), C. lusitaniae(n=3), and C. zeylanoides (n=1).

Clinical Specimens. A total of thirty-three yeast-positive blood culturebottles (FAN BacT/Alert, Organon Teknika) from routine testing at theClinical Microbiology Laboratory, Cleveland Clinic Foundation, OH wereincluded in this study. In addition, twenty-five simulatedyeast-positive blood culture bottles (FAN BacT/Alert, Organon Teknika)were made by inoculating routine blood culture bottles that werenegative following 7 days incubation. The inoculation was done with justa few colony-forming units of strains representing clinicallysignificant non-C. albicans species and comprised C. glabrata (n=2), C.lusitaniae (n=4), C. tropicalis (n=4), C. guilliermondii (n=1) C. krusei(n=3), C. parapsilosis (n=4), C. famata (n=2), C. norvegensis (n=4), andC. neoformans (n=1). These strains included reference strains from ATCCand the German Collection of Microorganisms and Cell. Cultures,Braunschweig, Germany (DSM) as well as recent clinical isolates from theInstitute of: Medical Microbiology, University Hospital RWTH Aachen,Aachen, Germany. The blood culture bottles were re-incubated in theBacT/Alert Microbial detection system (Organon Teknika) until they weredetected as positive by the system.

Preparation of Smears. One drop of phosphate-buffered saline (PBS) wasplaced in the well of a Teflon-coated microscope slide (Clear Coat, ErieScientific, Portsmouth, N.H.) and 10-25 μL of culture or a small dropwas added, mixed and spread throughout the well. The smear was fixed byeither placing the slide on an 80° C. slide warmer for 2 hours or at 60°C. for 20 minutes. The slide was subsequently immersed into 95% ethanolfor 1-2 minutes and allowed to air-dry.

Fluorescence In Situ Hybridization Using PNA Probes (PNA FISH). Smearswere covered with approximately 20 μL of hybridization solutioncontaining 10% (w/v) dextran sulfate (Sigma Chemical Co., St. Louis,Mo.), 10 mM NaCl, 30% (v/v) formamide (Sigma), 0.1% (w/v) sodiumpyrophosphate (Sigma), 0.2% (w/v) polyvinylpyrrolidone (Sigma), 0.2%(w/v) ficoll (Sigma), 5 mM Na₂EDTA (Sigma), 0.1% (v/v) Triton X-100(Aldrich), 50 mM Tris/HCl pH 7.5 and 250 nM fluorescein-labeled PNAprobe targeting C. albicans. Coverslips were placed on the smears toensure even coverage with hybridization solution, and the slides wereplaced on a slide warmer with a humidity chamber (Slidemoat, Boeckel,Germany) and incubated for 90 min at 55° C. Following hybridization, thecoverslips were removed by submerging the slides into approximately 20ml/slide pre-warmed 5 mM Tris, pH 10, 15 mM NaCl (J. T. Baker), 0.1%(v/v) Triton X-100 (Aldrich) in a water bath at 55° C. and washed for 30min. The slides were then air-dried. Each smear was finally mountedusing one drop of IMAGEN Mounting Fluid (DAKO, Ely, UK) and covered witha coverslip. Microscopic examination was conducted using a fluorescencemicroscope (Optiphot, Nikon Corporation, Tokyo, Japan) equipped with a60×/1.4 oil objective (Nikon), an HBO 100 W mercury lamp, and aFITC/Texas Red dual band filter set (Chroma Technology Corp.,Brattleboro, Vt.).

Results

The probe was tested on a panel of reference strains representing C.albicans and other Candida species, including phylogentically closelyrelated Candida species, clinically relevant Candida species and otheryeast species. The results are summarized in Table 2 and show that theprobe is highly specific.

TABLE 2 Results for reference strains analyzed by PNA FISH with C.albicans- specific PNA probe Yeast species Strain ID Can26S03/FluCandida albicans NRRL Y-107 + Candida albicans NRRL Y-12983 + Candidaalbicans NRRL Y-17967 + Candida albicans NRRL Y-17968 + Candida albicansNRRL Y-17974 + Candida albicans NRRL Y-17976 + Candida albicans NRRLY-302 + Candida albicans NRRL Y-477 + Candida albicans NRRL Y-6359 +Candida albicans NRRL Y-6943 + Candida albicans NRRL Y-79 + Candidaalbicans NRRL Y-81 + Candida albicans NRRL Y-82 + Candida albicans NRRLYB-3898 + Candida dubliniensis NRRL Y-17841 − Candida dubliniensis NRRLY-17512 − Candida dubliniensis NRRL Y-17969 − Candida dubliniensis NRRLY-17971 − Candida dubliniensis NRRL Y-17972 − Candida dubliniensis NRRLY-17973 − Candida glabrata NRRL Y 65 − Candida maltosa NRRL Y-17677 −Candida tropicalis NRRL Y-12968 − Candida tropicalis NRRL Y-1552 −Candida tropicalis NRRL Y-5716 − Candida viswanathii NRRL Y-6660 −Candida lodderae NRRL Y-17317 +/− Candida parapsilosis NRRL Y-12969 −Candida parapsilosis NRRL Y-543 − Candida sojae NRRL Y-17909 −Lodderomyces elongisporus NRRL YB-4239 − Lodderomyces elongisporus NRRLY-7681 − S. cerevisiae ATCC 4098 −

This is to our knowledge the first probe targeting rRNA that has beenshown not to react with C. dubliniensis, a recently discovered Candidaspecies that is often mis-identified as C. albicans using standardmethods, such as germ tube analysis and carbon assimilation methods.

The sensitivity and specificity of the C. albicans PNA FISH were furtherexamined using one hundred forty-eight clinical isolates representing C.albicans and other clinically relevant yeast species. The results aresummarized in Table 3. The assay correctly identified all C. albicansisolates and gave negative results with all other isolates.

TABLE 3 Reaction of C. albicans PNA FISH with 148 isolates representingclinically relevant yeast species. C. albicans PNA FISH PositiveNegative Species (n) (n) Candida albicans 72 0 Candida dubliniensis 0 58Candida glabrata 0 5 Candida tropicalis 0 3 Candida krusei 0 2 Candidaparapsilosis 0 4 Candida lusitaniae 0 3 Candida zeylanoides 0 1

The diagnostic performance of C. albicans PNA FISH was evaluateddirectly on 33 yeast-positive blood culture bottles as compared toresults obtained by standard methods. These comprised nine C. albicanscultures and twenty-four non-C. albicans cultures representing six toseven different species. The specificity was furthermore tested withtwenty-five simulated blood culture bottles representing nine differentspecies. The results are summarized in Table 4 and show 100% agreementwith standard methods supporting a 100% diagnostic sensitivity and 100%diagnostic specificity.

TABLE 4 Reaction of C. albicans PNA FISH with yeast-positive bloodcultures comprising 33 real blood culture bottles and 25 artificiallyspiked blood culture bottles. C. albicans PNA FISH Positive NegativeIdentification (n) (n) Candida albicans 9 0 Candida glabrata 0 11Candida parapsilosis 0 10 Candida tropicalis 0 5 Candida krusei 0 4Candida lusitaniae 0 9 Candida famata 0 2 Candida norvegensis 0 4Candida guilliermondii 0 1 Cryptococcus neoformans 0 2 Other yeast, notidentified 0 1Summary

PNA FISH using PNA probes targeting rRNA of C. albicans was used foridentification of C. albicans in yeast positive blood culture bottles ina time frame not possible using conventional methods. The test wasperformed on smears made directly from the blood culture bottles andinterpretation of results was conducted by microscopy, such that the PNAFISH procedure simply added the high specificity of PNA probes tostandard microbiological procedures (i.e. smear preparation andmicroscopy) to provide definitive identification.

The C. albicans specific PNA probe showed a very high degree ofspecificity, not only when tested with clinical isolates, but also whenfurther challenged with strains from the C. albicans clade. Thesefindings are ascribed to the high specificity of PNA probes combinedwith the use of the D1/D2 region of 26S rRNA as target; a region thathas been used for systematic studies of yeasts. These data are yetanother example of how molecular diagnostic methods using rRNA sequencesas target can replace classic phenotypically based microbiologicalidentification methods. In fact, several atypical C. albicans strainswere correctly identified by C. albicans PNA FISH.

C. albicans PNA FISH provides rapid and specific identification of C.albicans. No confirmation testing is required for identification, thusallowing the appropriate patient treatment to be administered morequickly, i.e. immediate treatment with fluconazole since almost allisolates of C. albicans are susceptible to this drug.

Example 2 PNA Probes that Distinguish Between C. dubliniensis and C.albicans

Probe sequence Can18S09/Flu Flu-O-EE-TAGCCAGAAGAAAGG-EE-NH₂(C. dubliniensis) Can26S05/Flu Flu-O-EE-ACAGCAGAAGCCGTG-EE-NH₂(C. albicans)

Note: E=compound having the formula:

Reference Strains. Reference strains were obtained from AgriculturalResearch Service Culture Collection (NRRL) Peoria, Ill. The strains werepropagated in yeast and mold broth (Difco Laboratories, Detroit, Mich.)at 30-35° C.

Preparation of Smears. One drop of phosphate-buffered saline (PBS) wasplaced in the well of a Teflon-coated microscope slide (Clear Coat, ErieScientific, Portsmouth, N.H.) and 10 μL of culture was added, mixed andspread throughout the well. The smear was fixed by either placing theslide on an 80° C. slide warmer for 2 hours or by flame-fixation bypassing the slides through the blue cone of a Bunsen burner. The slidewas subsequently immersed into 95% ethanol for 1-2 minutes and allowedto air-dry.

Fluorescence In Situ Hybridization Using PNA Probes (PNA FISH). Smearswere covered with approximately 10 μL of hybridization solutioncontaining 10% (w/v) dextran sulfate (Sigma Chemical Co., St. Louis,Mo.), 10 nM NaCl, 30% (v/v) formamide (Sigma), 0.1% (w/v) sodiumpyrophosphate (Sigma), 0.2% (w/v) polyvinylpyrrolidone (Sigma), 0.2%(w/v) ficoll (Sigma), 5 mM Na₂EDTA (Sigma), 0.1% (v/v) Triton X-100(Aldrich), 50 mM Tris/HCl pH 7.5 and 100 nM fluorescein-labeled PNAprobe targeting C. albicans or 500 nM fluorescein-labeled PNA probetargeting C. dubliniensis. Coverslips were placed on the smears toensure even coverage with hybridization solution, and the slides wereplaced on a slide warmer with a humidity chamber (Slidemoat, Boeckel,Germany) and incubated for 30 min at 50° C. Following hybridization, thecoverslips were removed by submerging the slides into approximately 20mL/slide pre-warmed 5 mM Tris, pH 10, 15 mM NaCl (J. T. Baker), 0.1%(v/v) Triton X-100 (Aldrich) in a water bath at 50° C. and washed for 30min. The slides were then air-dried. Each smear was finally mountedusing one drop of IMAGEN Mounting Fluid (DAKO, Ely, UK) and covered witha coverslip. Microscopic examination was conducted using a fluorescencemicroscope (Optiphot, Nikon Corporation, Tokyo, Japan) equipped with a60×/1.4 oil objective (Nikon), an HBO 100 W mercury lamp, and aFITC/Texas Red dual band filter set (Chroma Technology Corp.,Brattleboro, Vt.).

Results

Fifteen C. albicans and six C. dubliniensis reference strains weretested with the PNA FISH assay The results are summarized in Table 5. Ofthe fifteen C. albicans strains, 15 (100%) produced a positive resultwith the C. albicans PNA probe and a negative result with the C.dubliniensis PNA probe. Of the six C. dubliniensis strains, six (100%)produced a negative result with the C. albicans PNA probe and a positiveresult with the C. dubliniensis PNA probe.

TABLE 5 Results for reference strains analyzed by PNA FISH with C.albicans and C. dubliniensis PNA probes Organism Strain ID Can18S05/FluCan23S09/Flu C. albicans NRRL Y-12983 + − C. albicans NRRL Y-79 + − C.albicans NRRL Y-81 + − C. albicans NRRL Y-82 + − C. albicans NRRLY-107 + − C. albicans NRRL Y-302 + − C. albicans NRRL Y-477 + − C.albicans NRRL Y-6359 + − C. albicans NRRL Y-6943 + − C. albicans NRRLY-17967 + − C. albicans NRRL Y-17968 + − C. albicans NRRL Y-17974 + − C.albicans NRRL Y-17975 + − C. albicans NRRL Y-17976 + − C. albicans NRRLYB-3898 + − C. dubliniensis NRRL Y-17841 − + C. dubliniensis NRRLY-17512 − + C. dubliniensis NRRL Y-17969 − + C. dubliniensis NRRLY-17971 − + C. dubliniensis NRRL Y-17972 − + C. dubliniensis NRRLY-17973 − +Summary

C. dubliniensis shares many phenotypical characteristics with C.albicans and is therefore incorrectly identified as C. albicans bycurrent standard methods, such as germ tube analysis and commerciallyavailable carbon assimilation tests. We have here shown that PNA FISHusing PNA probes targeting rRNA of C. dubliniensis and C. albicans is a100% accurate method for the differentiation of C. albicans and C.dubliniensis.

Example 3 Analysis of a PNA Probe Specific to Candida glabrata

PNA Probe sequence Can18S11/Flu Flu-O-TCCAAGAGGTCGAGA-NH₂ EuUni/Cy3Cy3-OO-ACC-AGA-CTT-GCC-CTC-NH₂ Note: Cy3 = the cyanine 3 dye availablefrom Amersham Pharmacia Biotech.

Reference Strains. Reference strains were obtained from AgriculturalResearch Service Culture Collection (NRRL) Peoria, Ill. and AmericanType Culture Collection ATCC), Manassas, Va. The strains were propagatedin yeast and mold broth (Difco Laboratories, Detroit, Mich.) at 30-35°C.

Clinical Specimens. A total of seventeen yeast-positive blood culturebottles (FAN BacT/Alert, Organon Teknika) from routine testing at theClinical Microbiology Laboratory, Cleveland Clinic Foundation, OH wereincluded in this study.

Preparation of Smears. One drop of phosphate-buffered saline (PBS) wasplaced in the well of a Teflon-coated microscope slide (Clear Coat, ErieScientific, Portsmouth, N.H.) and 10 μL of culture was added, mixed andspread throughout the well. The smear was fixed by either placing theslide on an 80° C. slide warmer for 2 hours or at 60° C. for 60 minutes.The slide was subsequently immersed into 95% ethanol for 1-2 minutes andallowed to air-dry.

Fluorescence In Situ Hybridization Using PNA Probes (PNA FISH). Smearswere covered with approximately 5-10 μL of hybridization solutioncontaining 10% (w/v) dextran sulfate (Sigma Chemical Co., St. Louis,Mo.), 10 mM NaCl, 30% (v/v) formamide (Sigma), 0.1% (w/v) sodiumpyrophosphate (Sigma), 0.2% (w/v) polyvinylpyrrolidone (Sigma), 0.2%(w/v) ficoll (Sigma), 5 mM Na₂EDTA (Sigma), 0.1% (v/v) Triton X-100(Aldrich), 50 mM Tris/HCl pH 7.5 and 250 nM fluorescein-labeled PNAprobe targeting C. glabrata and 1 nM Cy3-labeled PNA probe targetingeukarya. EuUni/Cy3 was added in low conc. to give non-detected cells areddish appearance. Coverslips were placed on the smears to ensure evencoverage with hybridization solution, and the slides were placed on aslide warmer with a humidity chamber (Slidemoat, Boeckel, Germany) andincubated for 90 min at 55° C. Following hybridization, the coverslipswere removed by submerging the slides into approximately 20 mL/slidepre-warmed 5 mM Tris, pH 10, 15 mM NaCl (J. T. Baker), 0.1% (v/v) TritonX-100 (Aldrich) in a water bath at 55° C. and washed for 30 min. Theslides were then air-dried. Each smear was finally mounted using onedrop of IMAGEN Mounting Fluid (DAKO, Ely, UK) and covered with acoverslip. Microscopic examination was conducted using a fluorescencemicroscope (Optiphot, Nikon Corporation, Tokyo, Japan) equipped with a60×/1.4 oil objective (Nikon), an HBO 100 W mercury lamp, and aFITC/Texas Red dual band filter set (Chroma Technology Corp.,Brattleboro, Vt.).

Results

The probe was tested on a panel of reference strains representing C.glabrata and other Candida species, including phylogentically closelyrelated Candida species, clinically relevant Candida species and otheryeast species. The results are summarized in Table 6 and show that theprobe is highly specific.

TABLE 6 Results for reference strains analyzed by PNA FISH with C.glabrata- specific PNA probe Yeast species Strain ID Can18S11/FluCandida glabrata NRRL Y 65 + Candida glabrata NRRL Y-2242 + Candidaglabrata NRRL YB-3659 + Candida glabrata NRRL YB-3660 + Candida glabrataNRRL YB-4389 + Candida glabrata NRRL YB-4319 + Candida glabrata NRRLYB-1333 + Candida glabrata NRRL Y-1418 + Candida glabrata NRRL YB-4018 +Candida albicans NRRL Y-17968 − Candida albicans NRRL Y-17976 − Candidaalbicans NRRL Y-302 − Candida albicans NRRL Y-79 − Candida albicans NRRLY-81 − K. delphensis NRRL Y-2379 − K. bacillisporus NRRL Y-17846 − S.cerevisiae ATCC 4098 −

The diagnostic performance of C. glabrata. PNA FISH was evaluateddirectly on seventeen yeast-positive blood culture bottles as comparedto results obtained by standard methods. These comprised four C.glabrata cultures and thirteen non-C. glabrata cultures representingfour to five different species. The results are summarized in Table 7and show 100% agreement with standard methods supporting a 100%diagnostic sensitivity and 100% diagnostic specificity.

TABLE 7 Reaction of C. glabrata PNA FISH with seventeen routineyeast-positive blood cultures. C. glabrata PNA FISH Positive NegativeIdentification (n) (n) Candida albicans 0 8 Candida glabrata 4 0 Candidaparapsilosis 0 2 Candida tropicalis 0 1 Candida lusitaniae 0 1 Otheryeast, not identified 0 1

Having described preferred embodiments of the invention, it will nowbecome apparent to one of skill in the art that other embodimentsincorporating the concepts may be used. It is felt, therefore, thatthese embodiments should not be limited to disclosed embodiments butrather should be limited only by the spirit and scope of the invention.

We claim:
 1. A PNA probe set suitable for detecting, identifying orquantitating one or more species of Candida yeast in a sample, whereinthe probe set comprises PNA probes with probing nucleobase sequencesconsisting of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No. 1), AGA-GAG-CAA-CAT-GCA(Seq. Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id. No. 3),CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4), CAT-AAA-TGG-CTA-CCC-AG (Seq.Id. No. 5), ACT-TGG-AGT-CGA-TAG (Seq. Id. No. 6), CCA-AGG-CTT-ATA-CTC-GC(Seq. Id. No. 7), and CCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8), or theircomplements.
 2. The probe set of claim 1, wherein at least one probe ofthe set is unlabeled.
 3. The probe set of claim 1, wherein at least oneprobe is labeled with a detectable moiety.
 4. The probe set of claim 3,wherein the detectable moiety or moieties are selected from the groupconsisting of: a dextran conjugate, a branched nucleic acid detectionsystem, a chromophore, a fluorophore, a spin label, a radioisotope, anenzyme, a hapten, an acridinium ester and a chemiluminescent compound.5. The probe set of claim 1, wherein all the probes of the set arelabeled with one or more detectable labels.
 6. The probe set of claim 1,wherein two or more probes are independently detectable.
 7. The probeset of claim 1, wherein at least one probe of the set isself-indicating.
 8. The probe set of claim 1, wherein at least one probeof the set is support bound.
 9. A kit suitable for performing an assaythat determines the presence, absence or quantity of one or more speciesof Candida yeast in a sample, wherein said kit comprises: a) acombination of PNA probes, wherein the PNA probes comprise probingnucleobase sequences consisting of: AGA-GAG-CAG-CAT-GCA (Seq. Id. No.1), AGA-GAG-CAA-CAT-GCA (Seq. Id. No. 2), ACA-GCA-GAA-GCC-GTG (Seq. Id.No. 3) , CAT-AAA-TGG-CTA-CCA-GA (Seq. Id. No. 4) ,CAT-AAA-TGG-CTA-CCC-AG (Seq. Id. No. 5) , ACT-TGG-AGT-CGA-TAG (Seq. Id.No. 6) , CCA-AGG-CTT-ATA-CTC-GC (Seq. Id. No. 7) , andCCC-CTG-AAT-CGG-GAT (Seq. Id. No. 8), or their complements; and b) otherreagents or compositions necessary to perform the assay.
 10. The kit ofclaim 9, further comprising an antibody or antibody fragment whichspecifically binds to the PNA/nucleic acid complex.
 11. The kit of claim10, wherein the antibody is labeled with a detectable moiety.
 12. Thekit of claim 11, wherein the detectable moiety is selected from thegroup consisting of: a dextran conjugate, a branched nucleic aciddetection system, a chromophore, a fluorophore, a spin label, aradioisotope, an enzyme, a hapten, an acridinium ester and achemiluminescent compound.
 13. The kit of claim 9, further comprisingbuffers and/or other reagents suitable for performing a PNA-ISH orPNA-FISH assay.
 14. The kit of claim 9, wherein the PNA probes of thekit are unlabeled.
 15. The kit of claim 9, wherein at least one PNAprobe is labeled with a detectable moiety.
 16. The kit of claim 9,wherein two or more PNA probes are labeled with independently detectablemoieties.
 17. The kit of claim 16, wherein the independently detectablemoieties are independently detectable fluorophores.
 18. The kit of claim9, wherein at least one PNA probe is labeled with at least twoindependently detectable moieties.
 19. The kit of claim 9, furthercomprising buffers and/or other reagents suitable for performing anucleic acid amplification reaction.
 20. The kit of claim 9, wherein atleast one PNA probe is self-indicating.